Original research
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The Influence of Metal Salts,
Surfactants, and Wound Care Products
on Enzymatic Activity of Collagenase,
the Wound Debriding Enzyme
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Aleksa Jovanovic, PhD; Ryan Ermis, MPH; Rachel Mewaldt, BS;
Lei Shi, PhD; Dennis Carson, PhD, DABT
Abstract: An important part of the wound healing process is the removal
of necrotic tissue from a wound to promote healing. Enzymatic debridement is one of the widely used methods to accomplish this goal.
Clostridium collagenase (C. collagenase) containing ointment is frequently used in clinics to debride wounds. In this work, the influence
of metal salts and various types of surfactants on the enzymatic activity of C. collagenase is tested. The relationship between charge and
size of metal ions and surfactant structure is explained in the context
of enzyme inhibition. Commonly used wound care products, such as
cleansers, dressings, antimicrobial formulations, and silver dressings
are tested with C. collagenase. The results are discussed in terms of
enzyme compatibility with such materials, and recommendations for
use of wound care accessories in conjunction with the debriding enzyme are given, with the aim to help wound care providers make more
educated choices towards accomplishing optimal therapy outcome.
WOUNDS 2012;24(9):242–253
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Address correspondence to:
Lei Shi, PhD
Healthpoint Biotherapeutics
Research and Development
3909 Hulen Street
Fort Worth, TX 76107
[email protected]
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From Healthpoint Biotherapeutics,
Fort Worth, TX
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ffective wound debridement has been widely used to remove necrotic
tissue from a wound to promote healing. Necrotic tissue present in a
wound bed is undesirable because it prolongs the inflammatory stage
and may serve as a reservoir for bacterial growth, thus slowing the tissue
regranulation necessary for wound repair.1 It is increasingly well-recognized
that removal of nonviable tissue from a wound2 is an important step that
may facilitate the healing process for a variety of wound types, especially
burn wounds and various chronic wounds.3-5 Wound debridement may be
performed in several different ways: surgical, autolytic, enzymatic, and mechanical. Each of these has its own benefits and shortcomings, depending on
the wound type and the condition of the patient.6 7 Enzymatic debridement
can provide an effective methodology for various chronic ulcers, especially in
patient populations not amenable to surgical debridement.
Currently, Collagenase Santyl® Ointment (Healthpoint Biotherapeutics,
Fort Worth, TX) is the only Food and Drug Administration (FDA)-approved
enzymatic debriding biological in the United States.8 The enzyme activly used
in this drug is a bacterially derived collagenase from Clostridium histolyticum (C. collagenase). C. collagenase, a metalloproteinase with Zn2+ in the
Jovanovic et al
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the following: C. collagenase 0.1 mg/ml, FALGPA substrate
1 mg/ml, and the metal salts at concentrations varying
from 100-600 mM (refer to Table 1). The enzyme activity was measured as decreased absorbance at 345 nm for
the first 35 minutes at room temperature. The slope of
the linear curve is used as the activity rate (Vmax). In
the case of an insoluble salt, such as AgCl, the enzyme
solution was mixed with the powder for no less than 30
minutes, followed by centrifugation and the analysis of
the supernatant.
The activity of C. collagenase in the presence of surfactants was measured in a slightly different way.The concentrations of the enzyme, substrate, and surfactant were
as follows: C. collagenase 0.1 mg/mL, FALGPA 1 mg/ml,
and surfactant from 0.1-10 mg/mL (refer to Table 2). The
enzyme and surfactant containing solutions were incubated at room temperature for 30 minutes. The 5 millimolar
(mM) FALGPA solution was prepared in the assay buffer
(400 mM NaCl, 10 mM CaCl2, 50 mM Tricine, pH = 7.4),
and mixed well prior to use to ensure complete solubilization. Using a 96-well microplate, 100 L of the enzyme
solution was mixed with 150 L of the FALGPA solution.
The kinetic reaction was monitored for 35 minutes, and
kinetic rates (for enzymatic activity) were determined by
recording the absorbance change at 345 nm. Rates were
reported as Vmax milli-OD (1/1000 optical density unit)
per minute.The results for the metal salts and surfactants
are shown as percent enzymatic activity and are given by:
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active site, contains 2 principal enzyme species: collagenase I (Col H, 116 kDa, predominantly β-sheet structure)
and collagenase II (Col G, 124kDa, predominantly α helix
structure). Both enzymes specifically attack collagen in
wound necrotic tissue, which contains mostly the denatured collagens.9
Frequently, the enzymatic debrider is used in conjunction with various wound dressings10 to achieve multiple
treatment goals simultaneously, including infection control, pain control, and exudate management. Furthermore,
wound cleansers are often used before or even alongside
debriders to remove loosened tissue debris, bacteria, and
other physicochemical contaminants that can seriously
impede the wound healing process. Some dressings contain certain levels of metal elements (eg, silver) as principal bactericides, while wound cleansers rely on the cleaning power of various surfactants to remove the debris
from the wound bed.The purpose of this work is to evaluate the influence of various metal salts and surfactants on
C. collagenase enzymatic activity. Moreover, commercially
available wound care accessories, such as cleansers, dressings, and antibacterial preparations, will also be tested for
compatibility with the enzyme.The outcome of this study
should help wound care professionals make appropriate
choices with regards to dressings and cleansers (ie, commonly used accessories in the treatment of hard-to-heal
wounds) used alongside an enzymatic debrider to ensure
the optimal therapy outcome.
Materials and Methods
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C. collagenase was manufactured by Healthpoint Biotherapeutics (Fort Worth, TX). N-(3[2-Furyl]acryloyl)Leu-Gly-Pro-Ala (FALGPA), a chromogenic substrate, was
purchased from Bachem Americas, Inc (Torrance, CA). Poloxamers 124, 188, and 407 were gifts from BASF (Floram
Park, NJ). Cocamine oxide (Ammonyx®) was a gift from
Stepan (Northfield, IL). Cocamidopropyl dimonium chloride phosphate (Arlasilk™ PTC) was a gift from Croda, Inc
(Edison, NJ). Collagen FITC was purchased from Elastin
products Co, Inc. All other chemicals were purchased
from Sigma Aldrich (St. Louis, MO) and used without any
further purification.
All of the dressings, cleansers, and anti-bacterial accessories were purchased from the respective manufacturers or specialized stores (Tables 1-6).
Collagenase Activity Assays for Metal Salts and Surfactants. The collagenolytic activity of C. collagenase in
the presence of metal salts was measured using FALGPA.11
The concentrations of enzyme, substrate, and salts were
% Activity =
Vmax tested article
Vmax enzyme control solution
× 100
The results for influence of wound accessories on enzyme activity are shown as a percent inhibition and are
given by:
% Inhibition = 100 −
Vmax tested article
( Vmax enzyme control solution × 100 (
Collagenase activity assays for dressings and other
wound care products. The collagenolytic activity of C.
collagenase in presence of dressings was measured in
similar fashion for the metal salts and surfactants.The only
difference was that the dressing was soaked in the buffer
for at least 3 hours, and this buffer was then used to solubilize the enzyme.The measurement parameters were the
same as described above for salts and surfactants. Wound
cleansers that are water-based solutions (eg, ALLCLENZ®,
Healthpoint, Fort Worth,TX), were used as a solubilization
media for the enzyme. Antimicrobial powders (eg, PolyVol. 24, No. 9 September 2012
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Figure 2. Influence of transition metal salts on C. collagenase enzymatic activity.
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Figure 1. Influence of alkali and earth alkali metal chloride
salts on C. collagenase enzymatic activity.
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Figure 3. Influence of Na and Mg sulfates on enzymatic activity of C. collagenase.
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sporin®, Johnson and Johnson, New Brunswick, NJ) were
freely soluble in buffered solutions. Ointments, gel dressings, and creams (eg, Bactroban®, GlaxoSmithKline, Research Triangle Park, NC) were mixed with assay-buffered
solutions, followed by centrifugation and the analysis of
the supernatant. The measurement parameters were the
same as already described for salts and surfactants.
Collagenase activity assays for insoluble analytes.
Several testing articles (eg, Iodoform) were insoluble in
aqueous system, had similar absorption maximum as FALGPA substrate, and therefore could not be tested using
the FALGPA method. In this case, an alternative method
for determination of C. collagenase was employed: 20mg
of Collagen-FITC was dispersed in enzyme solution (positive control), buffer (negative control), analyte/enzyme
solution, and analyte solution alone. The samples were
incubated at RT for 90 minutes, followed by centrifugation and spectrofotometric analysis at 485 nm. The
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Figure 4. Influence of different concentrations of Na Ascorbate and Acetate on enzymatic activity of C. collagenase.
result is shown as percent inhibition and is given by:
% Inhibition = 100 −
Amax tested article
( Amax enzyme control solution × 100 (
Statistical Analysis
Enzyme activity with metal salts and surfactants data
were averaged from a group of 3 samples (n=3). Standard
deviation was calculated and displayed in the graphs. Enzyme inhibition data with wound care accessories were
averaged from a group of 3 samples (n=3) and displayed
in tables.
Results and Discussion
Influence of metal salts on enzymatic activity of C.
collagenase. It is well documented that certain metal
ions exhibit strong influence on the activity of metalloproteins.12,13 In this work, the influence of metal salts
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Jovanovic et al
Figure 5. Influence of different concentrations of Na2EDTA
on enzymatic activity of C. collagenase.
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(mostly chlorides) have been investigated on the enzymatic activity of C. collagenase. Moreover, salts with a
common cation (eg, sodium salts) having different anions
(eg, sulfate and acetate) were tested as well. The results
are summarized in Figures 1-5.
Chloride salts of alkali and earth alkali metals generally
exhibit either no or slightly positive effect on enzymatic
activity of C. collagenase (Figure 1). There is no apparent
difference between 100 mM and 500 mM concentrations.
It has been reported that NaCl markedly increases the
catalytic activity of thermolysin,14 a member of the metalloproteinase family.The reason behind this is that Kcat
increases due to favorable electrostatic interactions between the protein and the medium.15 Both of the alkali
metals (ie, Na and K) increased the activity of C. collagenase in a similar fashion.Therefore, it appears that the ion
size and hydration are not the only factors that contribute
to enzymatic activity increase.
It is known that enzymes of this family actually have
binding sites for divalent cations other than Zn2+, such as
Ca2+.These sites are responsible for the stabilization of the
enzyme structure. Therefore, as expected, Ca2+ enhanced
the activity of C. collagenase. A somewhat novel finding
is that ions smaller and bigger than Ca2+, Mg2+, and Ba2+
behave in a similar way. All 3 of the aforementioned metals have unoccupied d orbitals, unlike transition metals.
Transition metals, except Co2+ at lower concentrations,
exhibit a profound negative influence on enzymatic activity of C. collagenase (Figure 2). It is known that the
higher a concentration of Zn2+, although present in the
active site of the enzyme, diminishes the enzyme activity, probably due to steric hindrance of the active site (ie,
more than one Zn2+ is in the active site).16 All other metals,
Figure 6. Influence of non-ionic surfactants (Tween 20 and
Tween 80) on enzymatic activity of C. collagenase.
except for Ag+, were divalent cations and their influence
on enzymatic activity was compared to Ca2+.The main difference between the Ca2+ and the transition metal ions is
the population of the d orbitals. Ca2+ has empty d orbitals,
while transition metals have between 1 and 10 electrons
in these orbitals.The presence of such electrons gives rise
to a number of interesting properties such as paramagnetism and color. Moreover, d electrons can aid the complexation between the metal and the ligand (ie, enzyme),
which can lead to conformational changes of the latter
resulting in the decrease of enzymatic activity.17, 18
It is of special interest to investigate the influence of
Ag+ on C. collagenase since the Ag-based wound dressings
are often applied in conjunction with enzymatic wound
debriders in clinics.19 Ag+ is a monovalent cation like Na+
or K+; however, the difference between these metals is
the presence of d electrons in the case of Ag+. Similarly to
divalent cations, it was observed that metallic monovalent
ions with d electrons significantly inhibited the enzymatic activity of C. collagenase, compared to metals without
such electrons (ie, alkali metals). It is interesting to note
that silver inhibited the enzyme in the form of a soluble
cation (AgNO3), as well as in the form of an insoluble salt
Vol. 24, No. 9 September 2012
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Jovanovic et al
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Figure 7. The influence of polymeric non-ionic surfactants
(poloxamer 124 and poloxamer 188) on enzymatic activity
of C. collagenase.
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(AgCl). Various silver dressings will be discussed later.
We also tested the influence of metal salts (eg, Na+
and Mg2+) with a divalent anion, SO42-.The results showed
these salts have positive influence on enzymatic activity,
regardless of concentration (Figure 3).
Two salts commonly used in physiological buffers,
sodium acetate (NaAc), and sodium ascorbate (NaAsc),
were tested at 3 different concentrations (Figure 4). A
general trend towards an increase of enzymatic activity
was observed with the increase in concentration.
Another sodium salt commonly found in many wound
care products, disodium ethylenediaminetetraacetic acid
(EDTA), a strong metal chelating agent, was tested for enzyme compatibility. Results (Figure 5) suggest that EDTA
at concentrations > 0.1 mM (0.0037 w%) inhibits the activity of the enzyme. The reason for this behavior is the
complexation of metal ion (Zn2+) from the enzyme active
site by EDTA.20
Influence of surfactants on enzymatic activity of C.
collagenase. Based on the nature of their polar part (ie,
“head”), surfactants can be divided into 4 groups: non246
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Figure 8. The influence of cationic surfactants (CTC and
CEDB) on enzymatic activity of C. collagenase.
ionic, anionic, cationic, and zwitterionic.
Tween 20 and Tween 80 were chosen as representatives of small non-ionic surfactants. As shown in Figure
6, small non-ionic surfactants exhibited none to minimal
effect on the enzyme activity up to the concentration of
10 mg/mL.21 The concentration and physical state of the
surfactant, in the form of micelles (at > 0.1 mg/ml) or
individual molecules (at ≤ 0.1 mg/ml), seems to have no
influence on enzymatic activity.
Poly (ethylene oxide) - poly (propylene oxide) - poly
(ethylene oxide) (PEO-PPO-PEO) block copolymer-type
surfactant (pluronics or poloxamers), and were used as
models for polymeric non-ionic surfactants in this study
(Figure 7). It was found that these surfactants actually
have a positive effect on C. collagenase activity.22 This is
true for both surfactants tested, poloxamer 124 with hydrophilic-lipophilic balance (HLB) of 12-18, and the much
more hydrophilic poloxamer 188 with HLB of > 24. Furthermore, the positive influence was independent of concentration, at least in the range tested.This result appears
to be in line with the previous findings by Johnston et al23
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Figure 10. Influence of anionic surfactant (SDS) on enzymatic
Activity of C. collagenase.
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terionic surfactants, were the only class of surfactants that
displayed linear concentration-dependent inhibition of C.
collagenase (Figure 9). The linearity probably stems from
very low CMC. Therefore, the surfactant structure in water will not change with increasing concentration; only the
number of structures (ie, micelles) will change. Furthermore, due to their dual electrostatic nature (ie, both charges on the same molecule), they can easily bind to the enzyme and disrupt the physiological protein conformation.
Anionic surfactant, such as sodium dodecyl sulfate (SDS)
had minimal influence on enzymatic activity when it was
tested under the CMC (0.1 mg/ml). However, at CMC and
above, SDS completely inhibited the enzyme (Figure 10).
This behavior is expected since at least 1 C. collagenase
(Collagenase G) has predominantly an α-helix structure as
oppose to an SDS-resistant β-sheet.25 Moreover, as in the
case of cationic surfactants, charge interactions are the primary reason for the inhibition of enzymatic activity.
Influence of dressings and other wound care products
on enzymatic activity of C. collagenase. In this work, several classes of drugs and devices used frequently in wound
care settings have been tested for compatibility with C.
collagenase.These include silver, iodine, polymeric (eg, collagen), and other type dressings; wound cleansers; antimicrobial semi-solids (eg, creams and ointments); and antimicrobial actives (eg, gentamicin sulfate). (Refer to Tables 1
through 6).
Silver dressings are considered standard of care for treatment and prevention of infections in clinics today. In this
work the authors have tested several products containing
various forms of silver with C. collagenase. Generally, silver
products inhibit the enzymatic activity of C. collagenase,
as shown in Table 1. However, products that contain ionic
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Figure 9. The influence of amphoteric surfactants (Arlasilk
CDM and Ammonyx C) on enzymatic activity of C. collagenase.
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where poloxamer 407 had none to minimal influence on
enzymatic activity up to concentrations of 50 mg/ml. It is
likely that stabilizing and favorable solubilization effects
of a surfactant are responsible for the preservation and
enhancement of enzymatic activity.
Cationic surfactants tested in this study were cetyl
pyridinium chloride (CPC) and cetyl ethyl dimethyl ammonium bromide (CEDB). Surfactants of this type greatly
inhibit the enzyme activity (Figure 8), even at the lowest
concentration.24 Cationic surfactants are able to electrostatically bind to negatively charged amino acid residues
(acidic amino acids), those involved in the active site interacting with Zn2+ within the protein, and can also disrupt
the enzyme native conformation through hydrophobic interaction by their non-polar tail. It is interesting to note that
the inhibition happens at concentrations under the critical
micelle concentration (CMC) (0.1 mg/ml), persists at the
same level through 2 medium concentrations (0.5-1 mg/
ml), and finally completely prevails at the highest concentration (10 mg/ml).
Cocamidopropyl dimonium chloride phosphate (Arlasilk™ PTC) and cocamine oxide (Ammonyx®), the zwit-
Vol. 24, No. 9 September 2012
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Jovanovic et al
Table 1: Influence of silver dressings on enzymatic activity of C. collagenase.
Inhibition (%)
Type
Manufacturer
1
Silverlon
44.3
Silver dressing
Argentum Medical,
LLC
2
Acticoat
52.4
Silver dressing
Smith-Nephew
3
Silvadene (10%)
67.0
Silver dressing
Keltman
Pharmaceuticals
4
Silvasorb
25.0
Silver dressing
Medline
5
Algidex Ag
3.81
Silver dressing
DeRoyal
6
Maxorb Ag
18.0
Silver dressing
7
Mepilex Ag
15.7
Silver dressing
8
Aquacel Ag
7.7
9
Allevyn Ag
7.6
Description
TE
Product
Elemental Silver
Nanocrystalline silver
1% Silver sulfadiazine
in cream base
A
No
Ionic silver
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Ionic silver alginate
wound dressing
Ionic silver alginate
wound dressing
Molnlycke
Ionic silver silicone
dressing
Silver dressing
ConvaTec
Hydrofiber silver
dressing
Silver dressing
Smith & Nephew
Ionic silver urethane
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Medline
Table 2: Influence of iodine dressings on enzymatic activity of C. collagenase.
Product
Inhibition (%)
Type
10
Iodoflex
93.8
Iodine dressing
Smith-Nephew
Iodine dressing
11
Iodosorb
87.0
Iodine dressing
Smith-Nephew
Iodine gel
12
Iodoform
Iodine dressing
Invacare
Iodoform-impregnated
gauze
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1.6
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silver (Products 4-9, Table 1) in the form of water-insoluble
chloride salt tend to have a milder effect on the enzyme,
while products with elemental or nanocrystalline silver
(Products 1 and 2,Table 1) reduce the initial enzyme activity
to half. The product that contains silver ionically bound to
a sulfadiazine molecule (Product 3, Table 1) inhibits > 60%
of the initial activity of the enzyme. It appears the enzyme
inhibition of silver-containing products is a function of the
silver release potential (ie, available concentration) and
chemical reactivity of the various silver forms.26
Iodine containing products are also frequently used in
clinics to treat wound infections or for sole wound-cleaning applications. In this work, the authors have tested an
iodine dressing (Product 10, Table 2), an iodine-containing
gel (Product 11, Table 2), and iodine in the form of Iodoform (Product 12, Table 2). Both iodine-containing products almost completely inhibit the activity of the enzyme
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Manufacturer
Description
(Table 2). This finding is in line with previously observed
inhibition of matrix metalloproteinases (MMP) in chronic
wounds by Povidone-Iodine.27 However, iodine in the form
of Iodoform (Product 12, Table 2.) showed no inhibition
of C. collagenase, probably due to the fact that it is very
insoluble in the aqueous system.
The next set of products tested are various wound
dressings made of polymeric materials (Products 13-16, 2024, and 26-27, Table 3), semi-solid ointments (Products 17,
19, 25, and 28-31, Table 3), and porcine-derived extracellular matrix (ECM) (Product 18, Table 3). The results (Table 3) suggest these types of dressings generally will not
cause any inhibition of the enzymatic activity of C. collagenase. Few exceptions are Products 25 and Products 30-31,
which moderately to severely inhibit the activity of the enzyme. Product 25 inhibits the enzyme due to the presence
of poly (hexamethylene) biguanide (PHMB) that has great
Jovanovic et al
No
Product
Inhibition
(%)
Type
Manufacturer
13
Xeroform
0.0
Wound dressing
Kendall Healthcare
Bismuth in
petrolatum gauze
14
Procellera
5.8
Wound dressing
Vomaris
Wound dressing
15
Fibracol
2.7
Wound dressing
Systagenix
Collagen-based
Alginate dressing
16
Hydrofera
0.0
Wound dressing
Hydrofera LLC
Dye-containing
bacteriostatic dressing
17
Proshield
0.0
Wound dressing
Healthpoint
Biotherapeutics
PEG-based semi-solid
dressing
18
Oasis
6.1
Wound dressing
Healthpoint
Biotherapeutics
SIS product
19
Multidex Gel
< 5.0
Wound dressing
DeRoyal
Maltodextrin gel
dressing
20
Aquasorb
0.0
Wound dressing
DeRoyal
Hydrogel-polyurethane
dressing
21
Multipad
0.0
Wound dressing
DeRoyal
Non-adherent dressing
22
Covaderm
0.0
Wound dressing
DeRoyal
Absorbent-adhesive
dressing
23
Sofsorb
0.0
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Table 3: Influence of various wound dressings on enzymatic Activity of C. collagenase.
Wound dressing
DeRoyal
Super Absorbent dressing
24
Transeal
0.0
Wound dressing
DeRoyal
Transparent film
dressing (polyurethane)
25
Prontosan Gel
(10%)
96.5
Wound dressing
B.Braun Medical
PHMB-containing gel
26
Allevyn
0.0
Wound dressing
Smith & Nephew
Hydrocellular dressing
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Description
Mepilex
10.4
Wound dressing
Molnlycke
Soft silicone foam dressing
28
Carrasyn Hydrogel
0.0
Wound dressing
Medline
Carbopol-based
hydrogel
29
Medihoney
22.3
Wound dressing
Derma Sciences
Leptospermum Honey
dressing
30
Glucan Pro Cream
3000
69
Wound dressing
Brennen Medical
Petrolatum-based
dressing
31
Bismuth Formic
Iodide (BFI)
75.9
Wound dressing
Numark
Laboratories
BFI in talc powder base
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potential to bind the metal ion at the active site, thus effectively diminishing the enzyme activity. Product 30, thick
hydrophobic ointment, contains anionic surfactants (ie, cetyl esters) that can significantly inhibit the enzyme (Figure
10.). Product 31 is a powder that contains iodide, a known
C. collagenase inhibitor.
Wound cleansers are formulations designated to remove
impurities, bacteria, and debris from the wound bed. Several representative examples of such formulations (Products 32-50,Table 4) were tested with C. collagenase. Generally these products will lower the enzyme activity due to
the presence of cleansing agents (ie, surfactants necessary
to clean the wound.) The enzyme is severely inhibited by
products that contain strong zwitterionic and/or anionic
Vol. 24, No. 9 September 2012
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Table 4: Influence of various wound cleansers on Eenzymatic Activity of C. collagenase.
No
Product
Inhibition
(%)
Type
Manufacturer
32
Allclenz
68.8
Wound dressing
Healthpoint
Biotherapeutics
Amphotheric surfactant-based
cleanser
33
Lactated Ringers
Solution pH 6.7
25.0
Wound dressing
Hospira Inc
Sodium lactate based-cleanser
34
Anasept Spray
0.0
Wound dressing
Anacapa T
echnologies
Sodium hypochlorite-based
cleanser
35
MicrocynRx Spray
0.0
Wound dressing
Oculus
36
Dakin's Solution
(4X diluted)
0.0
Wound dressing
Century
Pharmaceuticals
37
Microklenz
100.0
Wound dressing
Medline
38
3M Wound
Cleanser
76.2
Wound dressing
3M
Pyridoxine- and Zn-salts-based
cleanser
39
Biolex
0.0
Wound dressing
BARD
Poloxamine 908 and potassium
-sorbate based cleanser
40
Gentell Wound
cleanser
63.0
Wound dressing
Gentell
Amphotheric surfactant-based
cleanser
41
SAF-Clens AF
77.7
42
Seaclens
42.0
43
Restore
44
45
A
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Description
Sodium hypochlorite-based
cleanser
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cleanser
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Amphotheric surfactant-based
cleanser with benzethonium chloride
Convatec
Amphotheric surfactant-based
cleanser
Wound dressing
Coloplast
Non-ionic surfactant- and EDTAbased cleanser
35.18
Wound dressing
Hollister
Poloxamer 188 and alkyl parabenbased cleanser
Dermal Wound
Cleanser
52.0
Wound dressing
Smith & Nephew
Non-ionic surfactant and EDTA-based
cleanser with benzethonium chloride
Skintegrity
70.0
Wound dressing
Medline
Amphotheric surfactant- and EDTAbased cleanser
N
O
T
D
Wound dressing
Shur-Clens
0.0
Wound dressing
Convatec
Poloxamer188-based cleanser
47
CarraKlenz
100.0
Wound dressing
Medline
Anionic surfactant-based cleanser
48
VASHE wound
therapy
0.0
Wound dressing
Puricore
Hypochlorite-based cleanser
49
Clorpactin
20.9
Wound dressing
Guardian
Laboratories
Hypochlorite-based cleanser
50
Dermaklenz
84.3
Wound dressing
Dermarite
Industries LLC
Pyridoxine HCl- and alcohol-based
cleanser
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surfactants such as those found in Products 32, 37, 40-41,
45, and 47 (Table 4). As discussed earlier (Figure 9), these
types of surfactants can alter the structure of enzyme, thus
inhibiting its activity. Furthermore, several products (Products 42, 44, and 45;Table 4) contain EDTA, a strong chelating agent that removes metal (ie, Zn2+) from the active site
of the enzyme. Wound cleansers that are formulated with
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antibacterial actives of cationic surfactant structure, such
as Products 37 and 44, also inhibit the enzymatic activity
as noted earlier (Figure 8). Products 33 and 38, although
surfactant free, inhibit enzyme activity due to a lower-thannecessary pH value for optimal enzymatic performance of
C. collagenase (pH = 6.6 versus pH =7 .2-7.6).28 Poloxamerbased wound cleansers, (eg, Products 39, 43, and 46, Ta-
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Table 5: Influence of various antibacterial formulations on enzymatic activity of C. collagenase.
Product
Inhibition (%)
Type
Manufacturer
Description
51
Bactroban Cream
0.0
Antibacterial dressing
Glaxo Smith Kline
52
Sulfamylon Cream
0.0
Antibacterial dressing
UDL Laboratories
53
Gentamycin
Sulfate Ointment
13.0
Antibacterial dressing
E.Fougera Co.
54
Kerlix Gauze
(PHMB)
100.0
Antibacterial dressing
Kendall Healthcare
Bacteriostatic gauze
55
Anasept Gel
83.0
Antibacterial dressing
Anacapa
Technologies
Wound Dressing
56
Clindamycin
Phosphate Gel
0.0
Antibacterial dressing
Greenstone LLC
Antibacterial hydrogel
TE
No
Antibacterial cream
Antibacterial cream
IC
A
Antibacterial cream
No
Product
Inhibition (%)
57
Mafenide Acetate
0.0
58
Polysporine
1.2
59
Mupirocin
0.0
60
Gentamycin Sulfate
61
Silver sulfadiazine
62
Chlorhexidine Gluconate
PL
Table 6: Influence of various antimicrobial actives on enzymatic activity of C. collagenase.
Type
Manufacturer
Description
Sigma Aldrich
Sulfonamide-based
antimicrobial
Antibacterial active
Johnson and Johnson
Protein antimicrobial
mixture
Antibacterial active
Sigma Aldrich
Carbohydrate-based
antimicrobial
9.8
Antibacterial active
DPT
Carbohydrate-based
antimicrobial
51
Antibacterial active
Sigma Aldrich
Silver-containing
antimicrobial
Antibacterial active
Sigma Aldrich
Biguanide-based/
antimicrobial
T
D
U
Antibacterial active
N
O
0.0
Benzalkonium
Chloride
99.0
Antibacterial active
Sigma Aldrich
Cationic surfactant
antimicrobial
64
p-DADMAC (from
Bioguard gauze)
0.0
Antibacterial active
Sigma Aldrich
Bacteriostatic polymer
65
Neomycin
0.0
Antibacterial active
ICN
Carbohydrate-based
antimicrobial
66
Metronidazole
100.0
Antifungal Active
DPT
Antifungal agent
O
63
D
ble 4), are generally very compatible with C. collagenase,
as noted earlier (Figure 7). The exception is Product 43,
which significantly inhibits the enzyme, probably due to
the high concentration of alkyl parabens present in the
formulation. Finally, Products 34-36 and 48-49, which are
based on sodium hypochlorite, are very compatible with C.
collagenase, with exception of powdered Product 49 that
contains unidentified residue after solubilization in water.
Antibacterial formulations (Products 51-56, Table 5)
generally exhibited minimal to no influence on the enzymatic activity of C. collagenase. Products 51-53 are emulsion creams with small molecule antimicrobials as actives,
product 56 is a hydrogel with antimicrobial active. None
of these formulations significantly inhibit C. collagenase,
with the exception of very mild inhibition by Product 53.
Product 54 is gauze impregnated with PHMB, a chemical
already described as a strong enzyme inhibitor (Product
25,Table 3). Product 55 is a sodium hypochlorite-based anVol. 24, No. 9 September 2012
251
Jovanovic et al
IC
A
TE
nature of the polar head. Non-charged surfactant molecules
generally tend to have minimal or even slightly positive influence on the enzymatic activity of C. collagenase. Large
non-ionics have a particularly positive effect on enzymatic
activity, probably due to favorable solubilization effects and
very mild surfactantcy that is unable to unfold the protein
structure. Charged surfactants, both cationic and anionic,
generally inhibit enzymatic activity even at the lowest concentration tested.This interesting difference was observed
in the behavior of these 2 types of surfactants: cationics
strongly inhibit the enzyme even at concentrations under
the CMC, but do not completely diminish the activity of
the enzyme until very high concentrations; anionics (eg,
SDS) exhibited only mild inhibition at concentrations under CMC, but completely attenuate the activity of the enzyme at any concentration above CMC.Amphoteric surfactants inhibit the activity of the enzyme proportionally with
their concentration.
An important part of this work was to evaluate the compatibility of various wound care products with C. collagenase. Most silver-containing products are not compatible
with the enzyme, significantly inhibiting its activity. However, certain products containing silver in ionic form, embedded in polymeric matrix or foam, exhibit only mild influence on enzymatic activity. Iodine-containing dressings
strongly inhibit enzymatic activity regardless of the form
(ie, foam dressing or gel). Commonly used wound dressings
are generally compatible with the enzymatic activity of C.
collagenase, leaving a plethora of choices for clinicians.
Wound cleansers tend to inhibit the enzymatic activity of
C. collagenase, mainly because of the presence of powerful
ionic or zwitterionic surfactants.The other reasons for the
enzyme inhibition are the presence of EDTA, inadequate
pH of the formulations, or presence of cationic surfactantlike structures in the formulas. Wound cleansers containing large block co-polymer surfactants (eg, poloxamers) or
formulas with sodium hypochlorite are compatible for use
with the collagenase debrider. If wound cleansers, for example, contain any other surfactant type, except non-ionic
or sodium hypochlorite salts, it is strongly advised to rinse
the cleanser from the wound bed with saline thoroughly
before applying the collagenase debrider. Antimicrobial
formulations as well as antimicrobial actives are generally
compatible with C. collagenase.
This work was performed with the intention to help
wound care professionals make more educated decisions
with respect to what type of supporting products should
be used along with a collagenase wound debrider.
Conclusions
T
D
U
PL
timicrobial gel that strongly inhibits the activity of the enzyme.This is somewhat contradictory with the earlier findings for Products 34-36 (Table 4), which contain the same
concentration of sodium hypochlorite, yet without any influence on enzyme activity. The difference is the presence
of a gelling component in Product 55, sodium magnesium
silicate, which is probably responsible for the inhibition of
the enzyme activity. Anti-fungal Product 66 (Table 6) completely inhibits the activity of the enzyme, probably due to
complexation of the metal by metronidazole (the active
molecule in the formula).
Finally, the authors tested drug actives commonly used
in antimicrobial formulations (Drug Substances 57-66, Table 6). Drug Substance 58 is a free-flowing white powder,
containing active peptide-based antimicrobials bacitracin
and polymyxin B in a lactose base. An important finding
is that these actives are compatible with C. collagenase
(Table 6). Products 59, 60, and 65 (Table 6), carbohydratebased antimicrobials, also have no influence on the enzymatic activity of C. collagenase. Drug Substance 61 (Table
6), a silver-based antimicrobial showed significant inhibitory effect (refer to Table 1). Drug Substances 57, 62, and
64 have no negative effect on the enzymatic activity of C.
collagenase, while cationic surfactant-like Drug Substance
63 strongly inhibited the activity of the enzyme. Product 66, an antifungal, exhibits the total inhibition of the
C.collagenase.
D
O
N
O
In summary, our results demonstrated the influence of
various metal salts and surfactants on the enzymatic activity of C. collagenase.Alkali metal salts are chemical entities
that generally have no to slight positive influence on enzymatic activity. It was noted that the positive effect on enzymatic activity is proportional to salt concentration, which
implies the electrostatic interactions between the protein
and salts are favorable for catalytic transformations. Earth
alkali metal salts also exhibited minimal or mild positive
influence on enzyme activity. However, divalent transitional metals strongly inhibit the enzymatic activity of C. collagenase. The main difference between the 2 populations
of divalent cations is the presence of electrons in the d
orbitals. Transitional metals have partially filled d orbitals,
and thus they are able to make donor-acceptor complexes
with the enzyme, change the structure of the latter, and
thus decrease its activity.The true nature of this interesting
phenomenon was far beyond the scope of this text.
The influence of surfactants on the enzymatic activity
of C. collagenase is primarily a function of the electrostatic
252
WOUNDS® www.woundsresearch.com
Jovanovic et al
Acknowledgements
16. The authors wish to thank Renée Carstens for medical
writing contributions.
bition dual effects of cobalt ion on thermolysin activity. J Biochem. 2007;141(6):843-853.
Reference
ase inhibitors. Biol Trace Elem Res.1998;64(1-3):197-213.
Clark RA. Cutaneous tissue repair: basic biologic considerations.
18. Mg(2+)-ATPase by metal ions and prevention and recovery of
Schultz GS, Sibbald RG, Falanga V, et al. Wound bed preparation:
inhibited activities by chelators. J Enzyme Inhib Med Chem.
2005;20(5):469-476.
A
a systematic approach to wound management. Wound Repair
19. Jain J, Arora S, Rajwade JM, Omray P, Khandelwal S, Paknikar
Regen. 2003;11 Suppl 1:S1-28.
3. Fowler E, van Rijswijk L. Using would debridement to help
KM. Silver nanoparticles in therapeutics: development of an
achieve the goals of care. Ostomy Wound Manage. 1995;41(7A
antimicrobial gel formulation for topical use. Mol Pharm.
2009;6(5):1388-1401.
Suppl):23S-35S.
4. Berger MM. Enzymatic debriding preparations. Ostomy Wound
5. 20. Elaboration of proteolytic enzymes in the presence and absence
Steed DL, Donohoe D, Webster MW, Lindsley L. Effect of ex-
of exogenous growth factors. Am J Pathol. 1995;146(1):210217.
PL
betic foot ulcers. Diabetic Ulcer Study Group. J Am Coll Surg.
21. Malek Alkasrawi, Eriksson T, Borjesson J, et al. The effect of
Tween-20 on simultaneous saccharification and fermentation
1996;183(1):61-64.
of softwood to ethanol. Enzyme and Microbial Technology.
Wright J, Shi L. Accuzyme® papain urea debriding ointment: a
2003;33(1):71-79
historical review. Wounds. 2010;15 (Supp. 4), 2-12. Ref Type: Ge-
22.
Klasen HJ. A review on the nonoperative removal of necrotic
conversion of softwood lignocellulose by poly(ethylene glycol)
tissue from burn wounds. Burns. 2000;26(3):207-222.
addition. Enzyme and Microbial Technology. 2007;40(4):754762.
Shi L, Carson D. Collagenase Santyl ointment: a selective agent
D
8. for wound debridement. J Wound Ostomy Continence Nurs.
2009;37(6 Suppl):S12-16.
9. 23. T
tissue with an ananain-based enzyme-debriding preparation.
rats. J Cardiovasc Pharmacol. 1997;29(5):580-585.
24. ties and transport. Proc Inst Mech Eng H. 2007;221(2):153-160.
N
O
10. Dryburgh N, Smith F, Donaldson J, Mitchell M. Debride25. Eur J Oral Sci. 2000;108(4):327-334.
27. 2008;16(3):CD006214.
ity: resistance to sodium dodecyl sulfate suggests a central role
for rigidity and a bias toward beta-sheet structure. Biochemistry.
1997;34(1):49-54.
2004;43(35):11248-11254.
26. Shi L, Ermis R, Kiedaisch B, Carson D. The effect of various
wound dressings on the activity of debriding enzymes. Adv
Gerlach RF, de Souza AP, Cury JA, Line SR. Effect of lead, cadmium
Skin Wound Care. 2010;23(10):456-462.
O
and zinc on the activity of enamel matrix proteinases in vitro.
D
13. 14. 15. Manning M, Colon W. Structural basis of protein kinetic stabil-
Jackson RJ, Lim DV, Dao ML. Identification and analysis of a collagenolytic activity in Streptococcus mutans. Curr Microbiol.
12. Leung S, Gironella A,Trigo C, Bhushan A, Daniels CK, Lai JC. Cationic surfactants and other factors that affect enzymatic activi-
Wound Repair Regen. 1996;4(4):433-443.
ment for surgical wounds. Cochrane Database Syst Rev.
Johnston TP, Palmer WK. Effect of poloxamer 407 on the activity of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in
Skrabut EM, Hebda PA, Samuels JA, et al. Removal of necrotic
11. Johan Borjesson, Peterson R, Tjerneld F. Enhanced enzymatic
U
neric.
7. Varani J, Perone P, Inman DR, et al. Human skin in organ culture.
Manage. 1993;39(5):61-69.
tensive debridement and treatment on the healing of dia-
6. Krsti D,, Krinulovi K, Vasi V. Inhibition of Na+/K(+)-ATPase and
I. J Am Acad Dermatol. 1985;13:701-725.
IC
2. Duffy B, Schwietert C, France A, et al.Transition metals as prote-
TE
17. 1. Hashida Y, Inouye K. Kinetic analysis of the activation-and-inhi-
Eming SA, Smola-Hess S, Kurschat P, Hirche D, Krieg T, Smola H.
Mallya SK,Van Wart HE. Inhibition of human neutrophil collage-
A novel property of povidon-iodine: inhibition of excessive pro-
nase by gold(I) salts used in chrysotherapy. Biochem Biophys
tease levels in chronic non-healing wounds. J Invest Dermatol.
2006;126(12):2731-2733.
Res Commun. 1987;144(1):101-108.
Inouye K, Kuzuya K, Tonomura B. Sodium chloride enhances
28. Shi L, Ramsay S, Ermis R, Carson D. pH in the bacteria-contam-
markedly the thermal stability of thermolysin as well as its cata-
inated wound and its impact on clostridium histolyticum col-
lytic activity. Biochim Biophys Acta. 1998;1388(1):209-214.
lagenase activity: implications for the use of collagenase wound
Inouye K, Kuzuya K,Tonomura B. Effect of salts on the solubility
debridement agents. J Wound Ostomy Continence Nurs.
of thermolysin: a remarkable increase in the solubility as well
2011;38(5):514-521.
as the activity by the addition of salts without aggregation or
dispersion of thermolysin. J Biochem. 1998;123(5):847-852.
Vol. 24, No. 9 September 2012
253